Aguilera-Romero, AuxiliadoraSabido-Bozo, SusanaLopez, SergioCortes-Gomez, AlejandroRodriguez-Gallardo, SofiaPerez-Linero, Ana MariaRiezman, IsabelleRiezman, HowardMuñiz, Manuel2022-10-312022-10-312021-08-13Aguilera-Romero A, Sabido-Bozo S, Lopez S, Cortes-Gomez A, Rodriguez-Gallardo S, Perez-Linero AM, et al. Determination of the lipid composition of the GPI anchor. PLoS One. 2021 Aug 13;16(8):e0256184http://hdl.handle.net/10668/4314In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.enAtribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/LipidsGlycosylphosphatidylinositolsMembranesCeramidesGenes, reporterLípidosGlicosilfosfatidilinositolesMembranasCeramidasGenes informadoresMedical Subject Headings::Anatomy::Cells::Cellular Structures::Cell MembraneMedical Subject Headings::Chemicals and Drugs::Organic Chemicals::Amides::CeramidesMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::GPI-Linked ProteinsMedical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, ReporterMedical Subject Headings::Chemicals and Drugs::Carbohydrates::Glycoconjugates::Glycolipids::GlycosylphosphatidylinositolsMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Luminescent Proteins::Green Fluorescent ProteinsMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass SpectrometryMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane GlycoproteinsMedical Subject Headings::Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::SaccharomycesMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Fungal Proteins::Saccharomyces cerevisiae Proteinsresearch article34388214open access10.1371/journal.pone.02561841932-6203PMC8362999